1. Field of the Invention
This invention relates to an immortalized derivative porcine stem cell line, PICM-19H, capable of differentiating exclusively into hepatocyte cells expressing hepatocyte function, for example, inducible enzyme activity, such as cytochrome P450 (CYP450) activity; an immortalized derivative porcine stem cell line, PICM-19B, capable of differentiating exclusively into bile duct cells expressing bile duct cell function and forming a complete (confluent) cell monolayer of basolaterally polarized cells; a bioartificial liver device comprising either the PICM-19H cells or the PICM-19B cells or both, a method of using the PICM-19H and/or PICM-19B stem cell lines in a bioartificial liver device or support to alleviate liver dysfunction, a method of using the PICM-19H and/or PICM-19B stem cell lines in a screening assay to detect a compound or new chemical entity which inhibits or promotes an enzyme activity involved in the metabolism of xenobiotics in the liver and/or to detect a compound or new chemical entity which results in cytotoxicity, hepatotoxicity, or hepatic dysfunction due to the metabolism of xenobiotics and/or endogenous substrates in the liver; and a screening assay kit comprising PICM-19H cells and/or PICM-19B cells.
2. Description of the Relevant Art
Cell lines that possess in vivo-like hepatocyte functions are needed for the biological component of bioartificial liver devices that are currently in development (Strain and Neuberger. 2002. Science 295: 1005-1009; Chamuleau et al. 2005. Metab. Brain Dis. 20: 327-335). Tumor-derived cell lines, of human or animal origin, are without exception compromised in their liver functions, presumably because of their lack of normal differentiation and uncontrolled growth characteristics (Nyberg et al., 1994. Ann. Surg. 220(1): 59-67; Wang et al. 1998. Cell Transplant 7: 459-468; Kobayashi et al. 2003a. J. Artif. Organs. 6: 236-244; Kobayashi et al. 2003b. Keio J. Med. 52: 151-157; Rodriguez-Antona et al. 2002. Xenobiotida 32: 505-520; Filippi et al. 2004. J. Hepatol. 41: 599-605). Although new cell lines transfected with immortalizing transgenes are being developed and tested, there is no assurance that these cell lines won't suffer from similar problems for similar reasons (Hoekstra and Chamuleau. 2002. Int. J. Artif. Organs. 25: 182-191; Kobayashi et al. 2003b, supra). To date, most clinically tested bioartificial liver devices have used fresh or frozen porcine hepatocytes as the cell component in the device (Hoekstra and Chamuleau, supra; Demetriou et al. 2004. Ann. Surg. 239 (5): 660-670). While some efficacy in patient support has been achieved using these “liver-harvested” hepatocytes (Demetriou et al., supra), they are also compromised as cell components of bioartificial liver devices because the harvested hepatocyte cells rapidly die within the bioartificial liver device, and in addition, the cells can be under attack by the patient's preformed antibodies and complement factors, and further, such cell preparations are variable, and, therefore, are a potentially unsafe, cell source (Rodriguez-Antona et al., supra; Filippi et al., supra; Di Nicuolo et al. 2005. Xenotransplantation 2: 286-292).
Presently, most testing of new pharmacological and chemical agents in vitro for the purpose of investigating any adverse reactions with liver cells and liver cell function is performed with primary hepatocyte cultures, hepatocyte cell lines, or microsomal preparations derived from liver tissue or cells (Bertz and Granneman. 1997. Clin. Pharmaokinet. 32: 210-258; Yan and Caldwell. 2001. Curr. Top. Med. Chem. 1: 403-425; Vermeir et al. 2005. Expert Opin. Drug Metab. Toxicol. 1: 75-90). Microsomal preparations, while useful for some assessment, cannot be used to assess and predict cellular enzyme inductions or transport processes (Shimada et al. 1994. J. Pharmacol. Exp. Ther. 270: 414-423; Gómez-Lechón et al. 2004. Curr. Drug Metab. 5: 443-462). Fresh primary hepatocyte cultures can provide in vitro models of liver cellular function and can be prepared from a variety of species, including from specific disease state animal models (Guillouzo, A. 1998. Environ. Health Perspect. 106 (Suppl. 2): 511-532; Ulrichova et al. 2001. Toxicol. Lett. 125: 125-132; Gómez-Lechón et al., supra). However, even hepatocyte preparations of excellent quality are limited in their growth and survival in vitro, and this therefore necessitates the continual acquisition of new hepatocytes from source liver tissue (Guillouzo, supra; Hoekstra and Chamuleau, supra; Rodriguez-Antona et al., supra). Good quality human liver tissue is frequently in short supply and must always be handled as if potentially infectious (Guillouzo, supra; Hoekstra and Chamuleau, supra). Animal source liver tissue can be obtained in steady quantity and is usually not an infectious disease hazard, but even here, reproducibility problems may exist as a result of animal-to-animal genetic variation, animal health, nutritional status, and stress levels, and, perhaps most importantly, the cell culturist's skill in preparing the hepatocyte cell suspension (Guillouzo, supra; Di Nicuolo et al., supra).
To address these problems liver cell models based on hepatocyte cell lines that grow continuously, i.e., are functionally immortal, have been used. Unfortunately, immortal hepatocyte cell lines, human or otherwise, are functionally compromised as a result of their intrinsic character of unabated growth and lack of normal differentiation, and they are therefore poor model systems with which to measure normal hepatocyte metabolism; particularly the phase I and II enzymatic reactions and the cellular transport properties that are used as a basis for estimating in vivo toxicokinetics and pharmacokinetics (Guillouzo, supra; Hoekstra and Chamuleau, supra; Wilkening et al. 2003. Drug. Metab. Dispos. 31: 1035-1042; Yan and Caldwell, supra; Chandra and Brouwer. 2004. Pharm. Res. (NY) 21: 719-735). Thus, improved in vitro models for the prediction of in vivo liver biotransformation and toxicity are needed to enable faster biological evaluation of new chemical entities and to reduce controversial and costly animal testing (Bertz and Granneman, supra; Guillouzo, supra; Yan and Caldwell, supra; Chandra and Brouwer, supra).
Given the limitations of the in vitro liver cell models discussed above, it is generally accepted that a cell line that exhibits unlimited growth, and yet which differentiates normally, e.g., a liver stem cell line, would provide the best biological component for a cell based extracorporal bioartificial liver assistance device. For similar reasons, a liver stem cell line having such characteristics would also be the best in vitro model with which to conduct pharmacological and toxicological assessments of new chemical entities and would enable assessments that are standardized and repeatable.
Here, we describe the porcine liver stem cell lines of the invention, PICM-19H and PICM-19B, two derivative cell lines of the ARS-PICM-19 cell line, that fulfill these needs. The ARS-PICM-19 parental cell line and an artificial liver device comprising them have been patented in U.S. Pat. No. 5,532,156 and U.S. Pat. No. 5,866,420, respectively, and are hereby incorporated by reference in their entirety. One derivative cell line, the PICM-19H cell line, is capable of differentiating into hepatocytes and no longer exhibits the ability to differentiate and self-organize into multi-cellular bile ductules. The other cell line, PICM-19B, appears to spontaneously arise from the bile duct differentiating cells, but results in a unique cell phenotype, i.e., a dome-forming polarized epithelium, not seen within the parental ARS-PICM-19 cell line population.